Methylation markers and targeted methylation probe panel

A mixture comprising (a) an assay panel for enriching modified polynucleotides for detecting cancer and/or a type of cancer in a subject, and (b) converted cell-free DNA (cfDNA) molecules or amplification products thereof;

• wherein the converted cfDNA molecules comprise deaminated nucleotides obtained by treating cfDNA molecules with a deaminating agent;
• wherein the assay panel comprises at least 1,000 polynucleotide probes, wherein the at least 1,000 polynucleotide probes comprise at least 500 different pairs of probes comprising a first probe and second probe, wherein the first probe differs from the second probe and overlaps with the second probe by an overlapping sequence of at least 30 contiguous bases;
• wherein the polynucleotide probes of the at least 500 different pairs of probes are configured to collectively hybridize to a modified polynucleotide obtained by processing cell-free DNA (cfDNA) molecules derived from at least 500 different target genomic regions of the subject, or amplification products thereof;
• wherein the target genomic regions are human genomic regions;
• wherein at least one probe for each of the at least 500 different target genomic regions (i) comprises a length of at least 45 bases complementary to a sequence of the target genomic region or a modified sequence thereof produced by the processing, and (ii) does not have at least 90% complementarity to at least 45 contiguous bases of 20 or more off-target regions in a GRCh37/hg19 genome;
• wherein an off-target region does not comprise any of the at least 500 different target genomic regions;
• and
• wherein each of the at least 500 different target genomic regions comprises at least five methylation sites and are differentially methylated in cancer samples relative to non-cancer samples.